This invention is an improvement on currently established procedures for the detection of genetic diseases resulting from mutations and deletions in genomic DNA sequences. Prenatal diagnosis and carrier detection of many X-linked diseases are available via Southern analysis using full length cDNA clones. Unfortunately, there are several major limitations that prevent widespread and routine use of Southern analysis for diagnosis of genetic disease. In many of the X-linked diseases, the defective sequences are unknown and probes are unavailable. In other diseases, such as X-linked muscular dystrophy, there are multiple exons, at least 60, scattered over a large area of genomic DNA, approximately 2.4 million bases. The introns average 35 Kb in length. In the case of muscular dystrophy, at least 7-9 separate cDNA subclones are necessary for Southern blot analysis to resolve each exon-containing restriction fragment for hyplotype assignment or diagnosis of genomic alterations. Furthermore, Southern analysis is an expensive, tedious, and time-consuming technique that requires the use of radioisotopes, making it unsuitable for routine use in clinical laboratories.
An alternative to Southern analysis for mutation and deletion detection is the polymerase chain reaction (PCR) described by Mullis et al. in U.S. Pat. No. 4,683,195 which issued on Jul. 28, 1987 and by Mullis in U.S. Pat. No. 4,683,202 which issued on Jul. 28, 1987. With PCR, specific regions of a gene can be amplified up to a million-fold from nanogram quantities of genomic DNA. After amplification the nucleic acid sequences can be analyzed for the presence of mutant alleles either by direct DNA sequencing or by hybridization with allele-specific oligonucleotide probes. The PCR technique has proven useful in the diagnosis of several diseases including .beta.-thalassemia, hemophilia A, sickle cell anemia and phenylketonuria. Routine screening for genetic diseases and exogenous DNA sequences, such as virus, with PCR, has been limited by the ability to conduct tests for only a single sequence at a time. Screening for a plurality of possible DNA sequences requires a cumbersomely large number of separate assays, thus increasing the time, expense, and tedium of performing such assays. For example, in some diseases, such as Duchenne muscular dystrophy (DMD), PCR diagnosis has been limited since point mutations leading to DMD have not been identified. Approximately 60% of the cases of DMD are due to deletions. The other 40% are unknown at present, but probably involve mutations of the intron-exon splice sites or the creation of premature stop codons. Thus a large gene like the DMD gene must be screened with multiple assays.
In both U.S. Pat. Nos. 4,683,195 and 4,683,202, procedures are described for amplification of specific sequences. Both patents describe procedures for detecting the presence or absence of at least one specific nucleic acid sequence in a sample containing a mixture of sequences. Although the patents claim at least one sequence and state that multiple sequences can be detected, they do not provide an effective procedure for amplifying multiple sequences at the same time. In the examples, single sequences are amplified or multiple sequences are amplified sequentially. Adding primers for a second sequence is usually possible, but when primers for more than two sequences are added the procedure falls apart. The present application is an improvement on the PCR method and solves the problems encountered when primers for multiple sequences are reacted simultaneously. The present invention describes a procedure for simultaneous amplification of multiple sequences, and for the application of this multiplex amplification procedure in order to detect a plurality of deletions within the same gene or within multiple genes.
The procedures of the present application provide improved methods for the detection of deletions in hemizygous genes on the X and Y chromosomes. The procedures are effective in detecting genetic diseases caused by deletions on the X or Y chromosome, for example, DMD. They are also effective in detecting homozyous deletions and may be used to simultaneously screen for many possible homozygous or hemizygous deletions as long as parts of the appropriate genetic sequences are known. The procedure for multiplex amplification also enables simultaneous analysis of multiple genetic loci regardless of the presence or absence of deletions.